The imprinting of multiple binding agents such as antibodies and oligonucleotides on planar substrates in the form of spots or stripes facilitates the simultaneous monitoring of multiple analytes such as antigens and DNA in parallel (“multiplexed”) binding assays. The miniaturization of this array format for increasing assay throughput and studying binding kinetics are described, for example, in R. Ekins, F. W. Chu, Clin. Chem. 37, 955-967 (1991); E. M. Southern, U. Maskos, J. K. Elder, Genomics 13, 1008-1017 (1992). In recent years, this approach has attracted substantial interest particularly in connection with performing extensive genetic analysis, as illustrated in G. Ramsay, Nat. Biotechnol. 16, 40-44 (1998); P. Brown, D. Botstein, Nat. Genet. 21, 33-37 (1999); D. Duggan, M. Bittner, Y. Chen, P. Meltzer, J. M. Trent, Nat. Genet. 21, 10-14 (1999); R. Lipshutz, S. P. A. Fodor, T. R. Gingeras, D. J. Lockhart, Nat. Genet. 21, 20-24 (1999).
The principal techniques of array fabrication reported to date include: refinements of the original “spotting” in the form of pin transfer or ink jet printing of small aliquots of probe solution onto various substrates, as illustrated in V. G. Cheung, et al., Nat. Genet. 21, 15-19 (1999); sequential electrophoretic deposition of binding agents in individually electrically addressable substrate regions, as illustrated in J. Cheng, et al., Nat. Biotechnol., 541-546 (1998); and methods facilitating spatially resolved in-situ synthesis of oligonucleotides, as illustrated in U. Maskos, E. M. Southern, Nucleic Acids Res. 20, 1679-1684 (1992); S. P. A. Fodor, et al., Science 251, 767-773 (1991) or copolymerization of oligonucleotides, as illustrated in A. V. Vasiliskov, et al., BioTechniques 27, 592-606 (1999). These techniques produce spatially encoded arrays in which the position within the array indicates the chemical identity of any constituent probe.
The reproducible fabrication of customized arrays by these techniques requires the control of microfluidics and/or photochemical manipulations of considerable complexity to ensure consistent performance in quantitative assays. Microfluidic spotting to produce, in quantitatively reproducible fashion, deposited features of 100 μm diameter involves dispensing of nanoliter aliquots with tight volume control, a task that exceeds the capabilities of currently available fluid handling methodologies. In addition, exposure of binding agents to air during the deposition process, typically several hours' in duration, has uncontrollable impact on the molecular configuration and the accessibility of the binding agents in subsequent binding assays. In-situ array synthesis relies on a sequence of multiple masking and photochemical reaction steps which must be redesigned to accommodate any changes in array composition. Finally, assay performance must be assessed “in-situ” for each array subsequent to immobilization of binding agents, an aspect of array manufacturing which raises difficult quality control and implementation issues.